microRNA Review Series
J. Cell. Mol. Med. Vol 12, No 6A, 2008 pp. 2181-2188
Guest Editor: M. Ivan
MicroRNAs in embryonic stem cells and early embryonic
development
Louise C. Laurent
a
a, b,
*
Department of Reproductive Medicine, Division of Maternal Fetal Medicine, University of California, San Diego, CA, USA
b
Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA
Received: August 4, 2008; Accepted: September 11, 2008
•
•
•
•
Introduction
miRNAs in early embryogenesis
miRNAs in embryonic stem cells
Roles of specific miRNAs in early embryogenesis
• Roles of specific miRNAs in embryonic
stem cells
• miRNAs with common seed sequences
• Conclusion
Abstract
MicroRNAs (miRNAs) are involved in the regulation of a broad range of biological processes. There is an extensive literature on the roles
of miRNAs in metazoan development, from early embryogenesis to lineage commitment, and formation and maturation of cellular subtypes and complex tissues. This review will present the evidence that miRNAs are essential for the earliest stages of metazoan development, including establishment and maintenance of the pluripotent embryonic stem cells (ESCs) from which all foetal tissues arise.
We will also discuss specific miRNAs that have been associated with early embryogenesis and pluripotency. Finally, we will point out
evidence showing that there is strong and highly redundant miRNA regulation of a common set of target genes in the early stages of
embryonic development and in ESCs.
Keywords: embryonic stem cells • microRNAs • gene expression • epigenetic • embryonic development
Introduction
MicroRNAs (miRNAs) are small noncoding RNAs implicated in
regulation of a wide array of biological processes in plants and
metazoans, including metabolic pathways, development and many
disease processes including cancers. There is an extensive literature on the roles of miRNAs in organismal development, from early
embryogenesis to lineage commitment, and formation and maturation of cellular subtypes and complex tissues. In this review, we will
focus on the roles of miRNAs in the earliest stages of metazoan
development, and in the establishment and maintenance of pluripotent progenitor cells from which all foetal tissues arise.
miRNAs in early embryogenesis
It is likely that miRNAs are essential at the earliest stages of development, although there appears to be somewhat conflicting evi*
Correspondence to: Louise C. LAURENT,
Department of Reproductive Medicine,
Division of Maternal Fetal Medicine, University of California,
200 West Arbor Drive, San Diego, CA 92103-8433, USA.
dence on this point. It may be that there are some species-specific
differences in the roles of miRNAs in development. However, many
of the discrepancies can be attributed to differences in experimental design, the contribution of maternal proteins and maternal
miRNAs, and differences in the life cycles of specific organisms.
Many experiments have been performed by knocking out dicer
and dgcr8, two enzymes involved in miRNA biogenesis, in various
organisms. In the nematode C. elegans, dcr-1 ⫺/⫺ worms are
viable, but infertile, and have some partially penetrant developmental abnormalities, such as absent alae and a burst vulva phenotype [1, 2]. dicer1 ⫺/⫺ zebrafish hatch and appear normal for
the first week after fertilization, then start having decreased activity and growth arrest, and eventually die between 14 and 21 days
after fertilization [3]. Mouse embryos lacking dicer1 or dgcr8
expression displayed more severe defects than their nematode
and zebrafish counterparts. By embryonic day E7.5, there were
50% fewer than expected dicer1 ⫺/⫺ embryos, and the dicer1 ⫺/⫺
Tel.: 619-543-2384
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E-mail: llaurent@ucsd.edu
© 2008 The Author
Journal compilation © 2008 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
doi:10.1111/j.1582-4934.2008.00513.x
embryos that were observed were stunted and morphologically
abnormal, with conspicuous absence of expression of both Oct4
and brachyury, which suggests depletion of the pool of pluripotent
stem cells, as well as arrest of development prior to gastrulation
[4]. Mouse embryos deficient in zygotic dgcr8 were early embryonic lethal and showed gross morphological defects by E6.5 [5].
Although there is developmental arrest at quite different stages
in these three organisms (the adult stage in C. elegans, the larval
stage in the zebrafish and prior to gastrulation in the mouse),
impairments in development appear at similar times after fertilization, suggesting that the arrest may not be stage-specific, but
rather occurs when falling levels of maternally contributed dicer or
dgcr8 reach critical thresholds. Therefore, the organism with the
shortest life cycle, C. elegans, which develops from egg to adult in
2.5 days can progress to adulthood without production of new
dicer mRNA or protein. In contrast, the zebrafish has an embryonic period of 2 days and a larval stage lasting 27 days, and
arrests 5 days into the larval stage, whereas the mouse arrests at
day 6.5–7.5 out of its 21-day gestational period.
Evidence that maternally contributed dicer1 mRNA is functional for the first week of development, and that maternally contributed dicer1 protein and/or miRNAs are present for ~3 days was
provided by a study using microinjection of morpholinos into onecell stage zebrafish zygotes. Morpholino knockdown of dicer1
mRNA resulted in arrest at approximately 3 days after fertilization
and death at 7 days after fertilization [3]. Further evidence was
provided using a germline replacement method in zebrafish to
eliminate both maternal and zygotic dicer1. Germ cells from dicer1
⫺/⫺ embryos were transplanted into germ-cell-depleted wildtype embryos. Crossing these fish containing dicer1 ⫺/⫺ germ
cells resulted in embryos that lack both maternal and zygotic
dicer1, termed MZdicer embryos. These embryos had normal axis
formation and regionalization, but displayed growth and developmental defects starting from the first day, including defects in gastrulation, somitogenesis, and brain and heart development [6].
Interestingly, microinjection of members of the mir-430 family of
miRNAs, which are highly expressed in the wild-type early embryo
[7], into MZdicer embryos at the one-cell stage rescued brain morphogenesis, but not heart development [6].
The critical role of miRNAs starting from the very first cell division in the mouse was demonstrated by crossing mice with a conditionally floxed dicer1 allele with mice that carried the Cre recombinase under the control of the oocyte-specific Zp3 promoter. In
this way, animals that lacked dicer1 only in their oocytes were
generated. Fertilizing these dicer1-depleted oocytes with sperm
from wild-type males resulted in zygotes that were unable to
undergo their first cell division, demonstrating that maternal
dicer1 was necessary at the first cleavage stage [8].
miRNAs in embryonic stem cells
Embryonic stem cells (ESCs) are derived from the inner cell mass
of blastocyst-stage embryos. The cardinal features of ESCs are the
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capacity for indefinite self-renewal, pluripotence and a normal
karyotype. In the mouse, the definitive proof of pluripotence is
germline transmission, whereby progeny whose genetic material
is entirely of ESC origin are produced. Common assays used to
demonstrate pluripotence in hESCs include the formation of
embryoid bodies (EBs) in vitro and transplantation into immunocompromised mice to generate mature teratomas.
As on the organismal level, the functions of miRNAs in ESCs
have been probed using gene knockout strategies. Dicer1-null
mESCs continued to display typical mouse ESC morphology and
express the pluripotence marker Oct-4/Pou5f1, but were deficient
in centromeric silencing. Moreover, they had a slowed proliferation
rate, and were unable to differentiate, shown by the failure to contribute to the tissues of chimeric mice, form teratomas in immunodeficient mice, or generate EBs in suspension culture [9, 10].
Dgcr8 null mouse ESCs also showed preserved ESC morphology and expression of pluripotence markers, as well as a longer
doubling time and defects in EB and teratoma formation [5].
However, the defect in differentiation appeared to be more severe
in the dicer1 null cells than the dgcr8 null cells, because the dgcr8
null EBs arrested later in the differentiation process (16 days,
rather than 8), and did express some markers of differentiation,
albeit at low levels. This might have been due to the fact that
dicer1 is necessary for processing of both miRNAs and siRNAs,
whereas dgcr8 is specific to the miRNA biogenesis pathway.
These data suggest that while miRNAs promote (but are not
required for) the self-renewal of ESCs, they are essential for
pluripotence. Further evidence supporting this conclusion was
shown by reintroducing the dicer1 gene into dicer1-null mESCs,
which had previously been shown to be unable to differentiate,
and rescuing the ability to differentiate [9].
Roles of specific miRNAs in early
embryogenesis
Evidence that a small number of specific miRNAs are critical for
early embryonic development was provided by a series of experiments from the Plasterk laboratory. miRNA expression profiling of
zebrafish embryos at different stages of development [7] revealed
that the mir-430 family of miRNAs were highly expressed between
5 and 48 hrs after fertilization. Several members of the mir-430
family were able to rescue the brain morphogenesis defect when
injected at the one-cell stage into embryos deficient in maternal
and zygotic dicer [6]. It was also proposed that the mir-430 family contributes to clearance of maternal mRNAs, an important step
in the maternal-to-zygotic transition [6]. The mRNA expression
profiles of dicer-deficient embryos and dicer-deficient embryos
complemented with injected mir-430 were compared to identify
putative mir-430 target mRNAs. The set of identified mRNAs was
enriched both for mRNAs with mir-430 target sequences in their
3⬘UTRs and for maternally expressed mRNAs. There was also
evidence that the targeted mRNAs were destabilized by deadenylation.
© 2008 The Author
Journal compilation © 2008 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
J. Cell. Mol. Med. Vol 12, No 6A, 2008
Table 1 Summary of large-scale miRNA sequencing papers including mESCs and hESCs. The number of samples analysed, the species and
embryonic stem cell lines represented, the number of known miRNAs observed and the number of novel miRNAs found are shown. J1aza indicates J1 mESCs treated with the DNA methyltransferase inhibitor 5’-aza-deoxycytidine
Landgraf et al. 2007 [18]
Calabrese et al. 2007 [19]
Morin et al. 2008 [20]
Bar et al. 2008 [21]
#Samples
256
4
2
2
Species
Human, mouse, rat
Mouse
Human
Human
ESC samples
(# reads/sample)
Male mESC (1254)
Dicer ⫹/⫹ (57,834)
H9 hESC (6,147,718)
H1-Undiff (280,185)
Female mESC (335)
Dicer ⫺/⫺ (54,339)
H9 EB (6,014,187)
H1-Diff (143,124)
J1 (149,986)
J1aza (155,934)
Known miRNAs
340 human/303 mouse/205 rat
360
334
191
Novel miRNAs
12 human/23 mouse/14 rat
46
83
13
In Drosophila, temporal reciprocity of expression of the mir-309
family and their predicted target mRNAs across the maternal-tozygotic transition has been observed [11].
The zebrafish model has also been used to elucidate functional
targets of the mir-430 family. Nodal is a TGFb family member
that activates a signalling pathway involved in left-right axis determination and mesoderm induction (reviewed in [12]). The mir-430
family has been implicated in the tuning of Nodal activity by
down-regulating both the Nodal agonist squint and the Nodal
antagonist lefty [13].
Roles of specific miRNAs in embryonic
stem cells
miRNA profiling of mouse and human embryonic stem cells
(mESCs and hESCs) has been performed using a variety of methods, and has been used to identify candidate miRNAs that may be
important in establishing or maintenance of the pluripotent phenotype. Cloning and sequencing of novel miRNAs using standard
methods has been used to identify miRNA expressed in mESCs
[14] and hESCs [15]. These authors used qualitative Northern blot
to demonstrate differential expression of a subset of the cloned
miRNAs in ESCs. Two studies measured miRNA expression in
murine ESCs by qRT-PCR [16, 17]. In all four studies, two clusters
of miRNAs were found to be strongly up-regulated in ESCs (mir302, mmu-mir-290/hsa-mir-371/372/373).
There have been four papers describing large-scale small-RNA
sequencing studies including ESC samples (Table 1). Landgraf
et al. sequenced ~330,000 clones from 256 small RNA libraries
from a variety of mouse, rat and human cells and tissues, including two mESC lines [18]. The results of this study included the discovery of a small number of novel candidate miRNAs, as well as
evidence that each cell type expressed only a limited repertoire of
approximately 70 miRNAs. However, these findings might have
stemmed from the shallowness of the sequencing performed
(only ~1000 clones per library were sequenced). The low clone
counts also precluded the collection of statistically significant
expression information from this dataset, but the results were
largely consistent with the previous four studies described above.
Calabrese et al. sequenced four mESC samples, two from a
floxed Dicer mESC cell line before (Dicer ⫹/⫹) and after (Dicer
⫺/⫺) deletion of one of the Dicer catalytic domains, and two from
the J1 mESC line before (J1) and after (J1aza) treatment with the
DNA methyltransferase inhibitor 5-aza-deoxycytidine [19]. This
group found that approximately 70% of the small RNAs found in
the Dicer ⫹/⫹ and J1 samples were encoded in 6 genomic clusters, including the mir-290, mir-17, mir-467 clusters, an imprinted
cluster on chromosome 12, the mir-21 cluster and the mir-15b/16
cluster. Noting that the mir-17, mir-15b/16 and mir -290 clusters
had been associated with the cell cycle or oncogenesis, the
authors suggested that a major role for miRNAs in ES cells was in
cell cycle regulation. One drawback of this study was the absence
of non-ESCs for comparison.
Morin et al. performed high-throughput sequencing on a sample of undifferentiated hESCs and a sample of EBs differentiated
from hESCs [20]. Of the four large-scale sequencing studies, this
paper reports the greatest depth of sequencing. However, this
study shows the greatest discrepancy from all the other reports.
Many of the miRNAs found to be overexpressed in ESCs compared to differentiated cells in other studies were found to be
expressed more highly in the EBs than the undifferentiated hESCs
in this study. In addition, there were examples of miRNAs that
might be expected to be coordinately expressed (e.g. those
located in the same genomic cluster, or those that have similar
seed sequences) displaying opposing expression patterns. A possible explanation for these findings lies in the choice of EBs as the
comparison sample. The generation of EBs from ESCs is an
© 2008 The Author
Journal compilation © 2008 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
2183
unpredictable process, which results in a mixed population of variable undifferentiated, differentiating and differentiated cells in
varying ratios. The EBs analysed in this study were collected at
15 days, at which time there can still be many undifferentiated or
partially differentiated cells present. It is possible that these
authors have found an interesting pattern of miRNA expression
that is present in cells undergoing early differentiation, but further
studies that carefully dissect out the timecourse of miRNA expression in a homogeneous population of differentiating cells will need
to be done to discern whether or not this is the case.
The most recent sequencing paper, by Bar et al., performed
high-throughput sequencing on undifferentiated and spontaneously differentiated hESCs. Results of this study were consistent with most of the other expression studies, with members of
the mir-302, mir-17 and mir-519 clusters showing higher
expression in the undifferentiated sample than the differentiated
sample [21].
Two common limitations among these four sequencing studies
were insufficient depth of sequencing and lack of replication, with
a single library constructed and sequenced for each sample. Given
that the error rates seen in high-throughput sequencing sets can be
quite substantial, and that different miRNAs can display vastly
different copy numbers, ranging from one to tens of thousands of
copies per cell, the expression levels of relatively rare miRNA
species are not well measured until the number of reads is in the
millions, which was only achieved in the Morin et al. study. The
actual variance in the sequencing data cannot be estimated without
adequate technical and biological replication, which was not
achieved in any of the sequencing studies. Each group used a different method for assessing whether candidate mature miRNA
sequences represented ‘real’ miRNAs. These factors may explain
why the overlap between lists of novel miRNAs reported in these
four sequencing studies was so poor, with only a handful of
sequences in common.
A comprehensive microarray profiling analysis of miRNAs in
hESCs in the context of a variety of multipotent and differentiated
cell lines from our group [22] confirmed many of the results of previous studies, as well as introducing a number of new findings. It
was observed that the group of miRNAs up-regulated in hESCs was
enriched for oncogenic miRNAs, whereas the group of miRNAs
down-regulated in hESCs was enriched for tumour suppressor
miRNAs. A large cluster of miRNAs on chromosome 19 adjacent to
the mir-371/372/373 cluster containing 54 miRNAs across 96 kb,
as well as the small mir-302, mir-371/372/373, mir-17, mir-106a
and mir-106b clusters were significantly up-regulated in hESCs.
The chromosome 19 cluster had initially been described as a placenta-specific cluster [23]. This study also found that a large
imprinted cluster on chromosome 14 containing 57 miRNAs was
significantly down-regulated in hESCs. This cluster of miRNAs is
coordinately regulated with the upstream noncoding RNA
GTL2/MEG3 [24]. Down-regulation of this cluster of miRNA has
been associated with high tumour cell proliferation and poor survival in ovarian cancer patients, leading to the hypothesis that
members of this cluster are tumour suppressor genes [25]. The
homologous mouse chromosome 12 cluster was found to be
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highly expressed in the paper by Calabrese et al. discussed above,
but because there were no non-ESC comparison cells in that study,
it was not observed in that report that the expression of this cluster is actually lower in ESCs than in differentiated cells.
A recent report was published using ChIP-Seq to study chromatin occupancy of miRNA genes in mESCs [26]. Mapping of
H3K4me3, which binds preferentially to transcription initiation
sites, H3K36me3, which is a chromatin mark associated with transcriptional elongation was used to define the promoter regions
and infer the transcript lengths of the miRNA genes. Mapping of
the polycomb group protein Suz12, and the pluripotence-associated transcription factors Oct4/Pou5f1, Sox2, Nanog and Tcf3 was
also performed. Interestingly, the promoter regions of both the
pluripotence-associated miRNA genes (mir-290 cluster, mir-302
cluster, mir-92 cluster and let-7g) and the lineage-specific miRNA
genes (mir-9, mir-124a, mir-155 and mir-708) were co-occupied
by the pluripotence-associated transcription factors. However, the
lineage-specific miRNA gene promoters were also occupied by
Suz12. These findings are consistent with results from the same
group suggesting that the pluripotence-associated transcription
factors bind to promoters of genes that are highly differentially
expressed between pluripotent and differentiated cells, and that
binding of polycomb group proteins distinguishes the genes that
are repressed in pluripotent cells from those that are active in
pluripotent cells.
miRNAs with common seed sequences
Target specificity of miRNAs for cognate mRNAs is strongly
influenced by Watson-Crick base-pairing between a ‘seed’
sequence located at positions 2-8 in the mature miRNA
sequence and target sequences in the mRNA 3⬘UTR [27–29].
Eighteen out of 76 miRNAs significantly up-regulated in hESCs
share a dominant seed sequence. These miRNAs share at least 6
out of 7 nucleotides in the seed sequence. In humans, the
miRNAs containing this dominant seed sequence are located in
the mir-302 cluster, the mir-371/372/373 cluster, the chromosome 19 cluster, and three homologous clusters on chromosome 13 (the tumour-associated mir-17 cluster), chromosome 7
(the mir-106b cluster) and the X chromosome (the mir-106a
cluster) (Table 2).
The large miRNA cluster on chromosome 19 is primate-specific, and is interspersed with primate-specific Alu repeat elements. It appears that other organisms have independently experienced massive expansions of miRNAs containing the same dominant seed sequence. Examples include the Drosophila mir-430
family, which has been shown to be important in early embryogenesis, as discussed above, as well as a large cluster found on
mouse chromosome 2, containing multiple members of the mir467 family, which was first described in the report by Landgraf
et al. [18], and the mir-427 family in Xenopus tropicalis, of which
there are ~80 copies clustered in the genome [30]. As noted previously, the lack of depth of sequencing in the Landgraf et al. study
© 2008 The Author
Journal compilation © 2008 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
J. Cell. Mol. Med. Vol 12, No 6A, 2008
Table 2 Table of human, mouse, zebrafish and frog microRNAs with seed sequences sharing at least 6 out of 7 nucleotides. The miRNAs significantly up-regulated in hESCs are highlighted in blue. Mature miRNA sequences are shown, with positions 1, 2-8, 9 and 10-end denoted. The chromosomal locations of the homologous human miRNA cluster are indicated at the left, and the arm of the precursor hairpin encoding the mature
miRNA sequence is listed to the right of each sequence.
Species: human
chr 4
chr 7
chr 13
chr 19
X chr
Mature miRNA nucleotide number
miRNA name
1
2-8
9
10-end
arm
hsa-miR-302a
T
AAGTGCT
T
CCATGTTTTGGTGA
3⬘
hsa-miR-302b
T
AAGTGCT
T
CCATGTTTTAGTAG
3⬘
hsa-miR-302c
T
AAGTGCT
T
CCATGTTTCAGTGG
3⬘
hsa-miR-302d
T
AAGTGCT
T
CCATGTTTGAGTGT
3⬘
hsa-miR-106b
T
AAAGTGC
T
GACAGTGCAGAT
5⬘
hsa-miR-93
A
AAGTGCT
G
TTCGTGCAGGTAG
5⬘
hsa-miR-17-5p
C
AAAGTGC
T
TACAGTGCAGGTAGT
5⬘
hsa-miR-18a
T
AAGGTGC
A
TCTAGTGCAGATA
5⬘
hsa-miR-20a
T
AAAGTGC
T
TATAGTGCAGGTAG
5⬘
hsa-miR-372
A
AAGTGCT
G
CGACATTTGAGCGT
3⬘
hsa-miR-373
G
AAGTGCT
T
CGATTTTGGGGTGT
3⬘
hsa-miR-519a
A
AAGTGCA
T
CCTTTTAGAGTGTTAC
3⬘
hsa-miR-519b
A
AAGTGCA
T
CCTTTTAGAGGTTT
3⬘
hsa-miR-519c
A
AAGTGCA
T
CTTTTTAGAGGAT
3⬘
hsa-miR-519d
C
AAAGTGC
C
TCCCTTTAGAGTGT
3⬘
hsa-miR-519e
A
AAGTGCC
T
CCTTTTAGAGTGT
3⬘
hsa-miR-520a
A
AAGTGCT
T
CCCTTTGGACTGT
3⬘
hsa-miR-520b
A
AAGTGCT
T
CCTTTTAGAGGG
3⬘
hsa-miR-520c
A
AAGTGCT
T
CCTTTTAGAGGGTT
3⬘
hsa-miR-520d
A
AAGTGCT
T
CTCTTTGGTGGGTT
3⬘
hsa-miR-520e
A
AAGTGCT
T
CCTTTTTGAGGG
3⬘
hsa-miR-526b*
A
AAGTGCT
T
CCTTTTAGAGGC
3⬘
hsa-miR-106a
A
AAAGTGC
T
TACAGTGCAGGTAGC
5⬘
hsa-miR-18b
T
AAAGTGC
A
TCTAGTGCAGTTA
5⬘
hsa-miR-20b
C
AAAGTGC
T
CATAGTGCAGGTAG
5⬘
Species: mouse
chr 4
Mature miRNA nucleotide number
miRNA name
1
2-8
9
10-end
arm
mmu-miR-302a
T
AAGTGCT
T
CCATGTTTTGGTGA
3⬘
mmu-miR-302b
T
AAGTGCT
T
CCATGTTTTAGTAG
3⬘
mmu-miR-302c
A
AGTGCTT
C
CATGTTTCAGTGG
3⬘
mmu-miR-302d
T
AAGTGCT
T
CCATGTTTGAGTGT
3⬘
Continued
© 2008 The Author
Journal compilation © 2008 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
2185
Table 2 Continued.
chr 7
chr 13
chr 19
X chr
Species-specific
mmu-miR-106b
T
AAAGTGC
T
GACAGTGCAGAT
5⬘
mmu-miR-93
C
AAAGTGC
T
GTTCGTGCAGGTAG
5⬘
mmu-miR-17
C
AAAGTGC
T
TACAGTGCAGGTAG
5⬘
mmu-miR-18a
T
AAGGTGC
A
TCTAGTGCAGATAG
5⬘
mmu-miR-20a
T
AAAGTGC
T
TATAGTGCAGGTAG
5⬘
mmu-miR-290-3p
A
AAGTGCC
G
CCTAGTTTTAAGCCC
3⬘
mmu-miR-291a-3p
A
AAGTGCT
T
CCACTTTGTGTGC
3⬘
mmu-miR-291b-3p
A
AAGTGCA
T
CCATTTTGTTTGT
3⬘
mmu-miR-292-3p
A
AAGTGCC
G
CCAGGTTTTGAGTGT
3⬘
mmu-miR-294
A
AAGTGCT
T
CCCTTTTGTGTGT
3⬘
mmu-miR-295
A
AAGTGCT
A
CTACTTTTGAGTCT
3⬘
mmu-miR-106a
C
AAAGTGC
T
AACAGTGCAGGTAG
5⬘
mmu-miR-18b
T
AAGGTGC
A
TCTAGTGCTGTTAG
5⬘
mmu-miR-20b
C
AAAGTGC
T
CATAGTGCAGGTAG
5⬘
mmu-miR-105
C
CAAGTGC
T
CAGATGCTTGTGGT
5⬘
mmu-miR-467a
T
AAGTGCC
T
GCATGTATATGCG
5⬘
mmu-miR-467b
G
TAAGTGC
C
TGCATGTATATG
5⬘
mmu-miR-467c
T
AAGTGCG
T
GCATGTATATGTG
5⬘
mmu-miR-467d
G
AAGTGCG
C
GCATGTATATGCG
5⬘
Species: zebrafish
Mature miRNA nucleotide number
miRNA name
1
2-8
9
10-end
arm
chr 7
dre-miR-93
A
AAAGTGC
T
GTTTGTGCAGGTA
5⬘
chr 13
dre-miR-17a
C
AAAGTGC
T
TACAGTGCAGGTA
5⬘
dre-miR-18a
T
AAGGTGC
A
TCTAGTGCAGATA
5⬘
dre-miR-20a
T
AAAGTGC
T
TATAGTGCAGGTAG
5⬘
dre-miR-18b
T
AAGGTGC
A
TTTAGTGCAGATA
5⬘
dre-miR-18c
T
AAGGTGC
A
TCTTGTGTAGTTA
5⬘
dre-miR-20b
C
AAGGTGC
T
CACAGTGCAGGTAG
5⬘
dre-miR-430a
T
AAGTGCT
A
TTTGTTGGGGTAG
3⬘
dre-miR-430ab
A
AAGTGCT
A
TCAAGTTGGGGTAG
3⬘
dre-miR-430c
T
AAGTGCT
T
CTCTTTGGGGTAG
3⬘
dre-miR-430i
T
AAGTGCT
A
TTTGTTGGCGTAG
3⬘
dre-miR-430j
A
AAGTGCT
A
TCAAATTGGGGTA
3⬘
X chr
Species-specific
Species: frog
chr 7
Mature miRNA nucleotide number
miRNA name
1
2-8
9
10-end
arm
xtr-miR-93a
A
AAGTGCT
G
TTCGTGCAGGTAG
5⬘
xtr-miR-93b
A
AGTGCTG
T
TCGTGCAGGTAG
5⬘
Continued
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© 2008 The Author
Journal compilation © 2008 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
J. Cell. Mol. Med. Vol 12, No 6A, 2008
Table 2 Continued.
chr 13
X chr
Species-specific
xtr-miR-17-5p
C
AAAGTGC
T
TACAGTGCAGGTAGT
5⬘
xtr-miR-18a
T
AAGGTGC
A
TCTAGTGCAGATAG
5⬘
xtr-miR-20a
T
AAAGTGC
T
TATAGTGCAGGTAG
5⬘
xtr-miR-106
A
AAAGTGC
T
TATAGTGCAGGTAGA
5⬘
xtr-miR-18b
T
AAGGTGC
A
TCTAGTGCAGTTAG
5⬘
xtr-miR-20b
C
AAAGTGC
T
CATAGTGCAGGTAG
5⬘
xtr-miR-427
G
AAAGTGC
T
TTCTGTTTTGGGCG
3⬘
precluded rigorous expression analysis. However, there was a
trend toward higher expression of the mir-467 cluster of miRNAs
in mESCs and embryonic cells. It is clear that the numerous
miRNAs that contain this ESC-dominant seed sequence are
descended from at least two unrelated ancestral DNA sequences,
because some of them are encoded on the 5⬘ arm of the precursor hairpin, whereas others are encoded on the 3⬘ arm (Table 2).
Overlapping functions of the mir-106b cluster and the mir-17
cluster were demonstrated in the mouse. Deletion of the mir-106b
cluster did not result in any detectable phenotype, and deletion of
the mir-17 cluster resulted in neonatal lethality, with lung hypoplasia, ventricular septal defect and defects in B cell development
[31]. Double mutants lacking both the mir-106b cluster and the
mir-17 cluster displayed a more severe phenotype, with embryonic lethality at midgestation. It would be interesting to discover
whether additional deletions of the mir-106a cluster, the mir-290
cluster and the mir-467 cluster in mouse would lead to a yet more
pronounced phenotype.
High expression of miRNAs containing this dominant seed
sequence in several different types of highly proliferative cells,
including hESCs, mESCs, early embryonic cells, placental cells
and tumour cells in a variety of different organisms, suggests
that down-regulation of a common set of target mRNAs may
enhance the proliferative properties of many diverse cell types.
However, the variation in sequences outside the seed among the
specific miRNAs in this set (see Table 2) and in the relative
amounts of the specific miRNAs among different cell types
points to the likelihood that there will be quantitative and qualitative differences in the set of mRNAs targeted by these miRNAs
in different cells types.
Conclusion
miRNAs play key roles at several stages in metazoan development.
They are essential for early embryogenesis and cellular differentiation, and enhance proliferation and the maternal-to-zygotic transition,
although they do not appear to be required for these latter functions.
In short, they seem to be essential for differentiation, but not selfrenewal. This is consistent with the observation that miRNAs appear
to have arisen around the same time as multicellularity, when cellular
differentiation became a more central concern of the organism. Only
one unicellular organism, Chlamydomonas reinhardtii, has been
shown to produce miRNAs [32], and these miRNAs appear to be
quite different from those found in other organisms.
The possibility that one of the primary functions of miRNAs
is to effect changes in cell state leads us to the notion that
miRNAs might be used as tools to efficiently direct cellular differentiation. The ability to control the phenotypes of cells would
be very useful in the development of desired cell types for clinical and preclinical applications, such as cell therapies or
in vitro testing of pharmaceuticals. In addition, one could imagine the use of miRNAs directly as therapeutic agents, such as
signals to direct cancer cells into a nonproliferative terminally
differentiated state.
Acknowledgements
We would like to thank Dr. Jeanne F. Loring for advice and mentorship. The
author is supported by an NIH-NICHD K12 Women’s Reproductive Health
Research Career Development fellowship.
References
1.
2.
Ketting RF, Fischer SE, Bernstein E, Sijen
T, Hannon GJ, Plasterk RH. Dicer functions in RNA interference and in synthesis
of small RNA involved in developmental
timing in C. elegans. Genes Dev. 2001; 15:
2654–9.
Grishok A, Pasquinelli AE, Conte D, Li N,
Parrish S, Ha I, Baillie DL, Fire A,
3.
Ruvkun G, Mello CC. Genes and mechanisms related to RNA interference regulate
expression of the small temporal RNAs
that control C. elegans developmental timing. Cell. 2001; 106: 23–34.
Wienholds E, Koudijs MJ, van Eeden FJ,
Cuppen E, Plasterk RH. The microRNAproducing enzyme Dicer1 is essential for
© 2008 The Author
Journal compilation © 2008 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
4.
zebrafish development. Nat Genet. 2003;
35: 217–8.
Bernstein E, Kim SY, Carmell MA,
Murchison EP, Alcorn H, Li MZ, Mills
AA, Elledge SJ, Anderson KV, Hannon
GJ. Dicer is essential for mouse
development. Nat Genet . 2003; 35:
215–7.
2187
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
2188
Wang Y, Medvid R, Melton C, Jaenisch
R, Blelloch R. DGCR8 is essential for
microRNA biogenesis and silencing of
embryonic stem cell self-renewal. Nat
Genet. 2007; 39: 380–5.
Giraldez AJ, Cinalli RM, Glasner ME,
Enright AJ, Thomson JM, Baskerville S,
Hammond SM, Bartel DP, Schier AF.
MicroRNAs regulate brain morphogenesis
in zebrafish. Science. 2005; 308: 833–8.
Wienholds E, Kloosterman WP, Miska E,
Alvarez-Saavedra E, Berezikov E, de
Bruijn E, Horvitz HR, Kauppinen S,
Plasterk RH. MicroRNA expression in
zebrafish embryonic development. Science.
2005; 309: 310–1.
Tang F, Kaneda M, O’Carroll D, Hajkova P,
Barton SC, Sun YA, Lee C, Tarakhovsky A,
Lao K, Surani MA. Maternal microRNAs
are essential for mouse zygotic development. Genes Dev. 2007; 21: 644–8.
Kanellopoulou C, Muljo SA, Kung AL,
Ganesan S, Drapkin R, Jenuwein T,
Livingston DM, Rajewsky K. Dicer-deficient mouse embryonic stem cells are
defective in differentiation and centromeric
silencing. Genes Dev. 2005; 19: 489–501.
Murchison EP, Partridge JF, Tam OH,
Cheloufi S, Hannon GJ. Characterization
of Dicer-deficient murine embryonic stem
cells. Proc Natl Acad Sci USA. 2005; 102:
12135–40.
Bushati N, Stark A, Brennecke J, Cohen
SM. Temporal reciprocity of miRNAs and
their targets during the maternal-tozygotic transition in Drosophila. Curr Biol.
2008; 18: 501–6.
Shen MM. Nodal signaling: developmental
roles and regulation. Development. 2007;
134: 1023–34.
Choi WY, Giraldez AJ, Schier AF. Target
protectors reveal dampening and balancing of Nodal agonist and antagonist by
miR-430. Science. 2007; 318: 271–4.
Houbaviy HB, Murray MF, Sharp PA.
Embryonic stem cell-specific MicroRNAs.
Dev Cell. 2003; 5: 351–8.
Suh MR, Lee Y, Kim JY, Kim SK, Moon
SH, Lee JY, Cha KY, Chung HM, Yoon HS,
Moon SY, Kim VN, Kim KS. Human embryonic stem cells express a unique set of
microRNAs. Dev Biol. 2004; 270: 488–98.
Strauss WM, Chen C, Lee CT, Ridzon D.
Nonrestrictive developmental regulation of
microRNA gene expression. Mamm
Genome. 2006; 17: 833–40.
Chen C, Ridzon D, Lee CT, Blake J, Sun Y,
Strauss WM. Defining embryonic stem
18.
19.
20.
21.
22.
23.
24.
cell identity using differentiation-related
microRNAs and their potential targets.
Mamm Genome. 2007; 18: 316–27.
Landgraf P, Rusu M, Sheridan R, Sewer
A, Iovino N, Aravin A, Pfeffer S, Rice A,
Kamphorst AO, Landthaler M, Lin C,
Socci ND, Hermida L, Fulci V, Chiaretti
S, Foa R, Schliwka J, Fuchs U, Novosel
A, Muller RU, Schermer B, Bissels U,
Inman J, Phan Q, Chien M, Weir DB,
Choksi R, De Vita G, Frezzetti D,
Trompeter HI, Hornung V, Teng G,
Hartmann G, Palkovits M, Di Lauro R,
Wernet P, Macino G, Rogler CE, Nagle
JW, Ju J, Papavasiliou FN, Benzing T,
Lichter P, Tam W, Brownstein MJ, Bosio
A, Borkhardt A, Russo JJ, Sander C,
Zavolan M, Tuschl T. A mammalian
microRNA expression atlas based on small
RNA library sequencing. Cell. 2007; 129:
1401–14.
Calabrese JM, Seila AC, Yeo GW, Sharp
PA. RNA sequence analysis defines Dicer’s
role in mouse embryonic stem cells. Proc
Natl Acad Sci USA. 2007; 104: 18097–102.
Morin RD, O’Connor MD, Griffith M,
Kuchenbauer F, Delaney A, Prabhu AL,
Zhao Y, McDonald H, Zeng T, Hirst M,
Eaves CJ, Marra MA. Application of massively parallel sequencing to microRNA
profiling and discovery in human embryonic stem cells. Genome Res. 2008; 18:
610–21.
Bar M, Wyman SK, Fritz BR, Qi J, Garg
KS, Parkin RK, Kroh EM, Bendoraite A,
Mitchell PS, Nelson AM, Ruzzo WL, Ware
C, Radich JP, Gentleman R, RuoholaBaker H, Tewari M. MicroRNA discovery
and profiling in human embryonic stem cells
by deep sequencing of small RNA libraries.
Stem Cells. 2008; 26: 2496–505.
Laurent LC, Chen J, Ulitsky I, Mueller FJ,
Lu C, Shamir R, Fan JB, Loring JF.
Comprehensive microRNA profiling
reveals a unique human embryonic stem
cell signature dominated by a single seed
sequence. Stem Cells. 2008; 26: 1506–16.
Bentwich I, Avniel A, Karov Y, Aharonov
R, Gilad S, Barad O, Barzilai A, Einat P,
Einav U, Meiri E, Sharon E, Spector Y,
Bentwich Z. Identification of hundreds of
conserved and nonconserved human
microRNAs. Nat Genet. 2005; 37: 766–70.
Seitz H, Royo H, Bortolin ML, Lin SP,
Ferguson-Smith AC, Cavaille J. A large
imprinted microRNA gene cluster at the
mouse Dlk1-Gtl2 domain. Genome Res.
2004; 14: 1741–8.
25. Zhang L, Volinia S, Bonome T, Calin GA,
Greshock J, Yang N, Liu CG, Giannakakis
A, Alexiou P, Hasegawa K, Johnstone CN,
Megraw MS, Adams S, Lassus H, Huang
J, Kaur S, Liang S, Sethupathy P, Leminen
A, Simossis VA, Sandaltzopoulos R,
Naomoto Y, Katsaros D, Gimotty PA,
DeMichele A, Huang Q, Butzow R, Rustgi
AK, Weber BL, Birrer MJ, Hatzigeorgiou
AG, Croce CM, Coukos G. Genomic and
epigenetic alterations deregulate microRNA
expression in human epithelial ovarian
cancer. Proc Natl Acad Sci USA. 2008;
105: 7004–9.
26. Marson A, Levine SS, Cole MF, Frampton
GM, Brambrink T, Johnstone S, Guenther
MG, Johnston WK, Wernig M, Newman J,
Calabrese JM, Dennis LM, Volkert TL,
Gupta S, Love J, Hannett N, Sharp PA,
Bartel DP, Jaenisch R, Young RA.
Connecting microRNA genes to the core
transcriptional regulatory circuitry of
embryonic stem cells. Cell. 2008; 134:
521–33.
27. Krek A, Grun D, Poy MN, Wolf R,
Rosenberg L, Epstein EJ, MacMenamin
P, da Piedade I, Gunsalus KC, Stoffel M,
Rajewsky N. Combinatorial microRNA target predictions. Nat Genet. 2005; 37: 495–
500.
28. Lewis BP, Burge CB, Bartel DP.
Conserved seed pairing, often flanked by
adenosines, indicates that thousands of
human genes are microRNA targets. Cell.
2005; 120: 15–20.
29. Lewis BP, Shih IH, Jones-Rhoades MW,
Bartel DP, Burge CB. Prediction of mammalian microRNA targets. Cell. 2003; 115:
787–98.
30. Tang GQ, Maxwell ES. Xenopus
microRNA genes are predominantly
located within introns and are differentially
expressed in adult frog tissues via posttranscriptional regulation. Genome Res.
2008; 18: 104–12.
31. Ventura A, Young AG, Winslow MM,
Lintault L, Meissner A, Erkeland SJ,
Newman J, Bronson RT, Crowley D,
Stone JR, Jaenisch R, Sharp PA, Jacks T.
Targeted deletion reveals essential and
overlapping functions of the miR-17
through 92 family of miRNA clusters. Cell.
2008; 132: 875–86.
32. Molnar A, Schwach F, Studholme DJ,
Thuenemann EC, Baulcombe DC.
miRNAs control gene expression in the
single-cell alga Chlamydomonas reinhardtii. Nature. 2007; 447: 1126–9.
© 2008 The Author
Journal compilation © 2008 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd